The sensory receptor neurons of the visual and auditory systems transmit information about stimulus properties via synaptic outputs that are specialized for transmission of both fast, transient and slower, sustained signals. The hallmark of these specialized synapses is the presence at the active zone of a complex organelle, the synaptic ribbon, which enhances the size of the readily releasable pool of synaptic vesicles. Although vision and hearing are impaired when synaptic ribbons are disrupted, exactly how the ribbon supports neurotransmitter release has been unclear. Numerous synaptic vesicles are attached to the surface of the ribbon, and these vesicles are thought to support both the fast and sustained components of transmission during stimulation. Previously in this project, methods were developed to track single synaptic vesicles associated with the ribbon at super-resolution in living synapses during neurotransmitter release, and to detect when vesicles fuse to release their contents. The results showed that ribbons have the dual role of serving as a conduit for diffusion of tethered vesicles and as a scaffold that supports compound fusion of vesicles. In the proposed work, the novel direct-imaging approach will be used to quantify all aspects of the life cycle of synaptic vesicle at ribbon active zones, including capture by the ribbon, translocation, fusion, clearance, and recycling by endocytosis. Direct tests of the compound-fusion model will also be carried out using cryofixation electron microscopy of ribbon synapses rapidly frozen while in the process of release. An additional goal of the project is to determine the molecular mechanisms that govern vesicle trafficking at ribbon active zones, in order to provide a comprehensive understanding that integrates molecular and physiological views of synaptic function. The results of the project will lead to significant new information about fundamental cellular and molecular mechanisms that control the early steps in transmission of sensory information in both vision and hearing.